CN101603964A - The enzyme-linked immune detection method of Anisakid nematode in the marine product - Google Patents
The enzyme-linked immune detection method of Anisakid nematode in the marine product Download PDFInfo
- Publication number
- CN101603964A CN101603964A CNA2009100168209A CN200910016820A CN101603964A CN 101603964 A CN101603964 A CN 101603964A CN A2009100168209 A CNA2009100168209 A CN A2009100168209A CN 200910016820 A CN200910016820 A CN 200910016820A CN 101603964 A CN101603964 A CN 101603964A
- Authority
- CN
- China
- Prior art keywords
- sample
- anisakid nematode
- nematode
- anisakid
- detection method
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses the enzyme-linked immune detection method of Anisakid nematode in a kind of marine product, the somatic antigen of this method separation and purification Anisakis larva utilizes the biological immune technology to prepare specific polyclonal antibody; With 96 hole polystyrene plates is solid phase carrier, wraps by, carrier protein with corresponding complete antigen in advance and seals; Simultaneously with the negative contrast of the extract that does not contain determinand, set up typical curve during detection with the titer of Anisakid nematode antigen; Utilize the light absorption value of conventional indirect competitive enzyme-linked immunosorbent absorption (ELISA) test sample and calculate corresponding inhibition ratio, and then according to the concentration of determinand (in somatic antigen) in the standard curve determination sample.The present invention is highly sensitive, accuracy good; Easy and simple to handlely fast effectively shortened analysis time, the rapid screening that can be applicable to Anisakid nematode in the marine product such as fish detects.
Description
Technical field
The present invention relates to pathogenic parasitic detection method in a kind of detection marine product, specifically relate to a kind of enzyme-linked immune detection method that detects Anisakid nematode.The present invention builds special fund (nycytx-50-G09) subsidy by national 863 projects (No.2007AA091806) and modern agriculture industrial technology system and finishes.
Background technology
Anisakid nematode (anisakid larvae) is the different sharp section of an Ascaridata nematode, be to a class parasite of human body harmfulness maximum in the present marine product, its pathogenic stage main parasitic fish that dwell in the sea, at present pathogenic strong, distribute mainly be comparatively widely Anisakis nematode and puppet newly the ascarid line belong to nematode.Along with marine product is eaten the rise gradually of mode raw, this parasite very likely becomes one of important hazard factor that influences China's marine product edible safety from now on.
Detection for Anisakid nematode in the marine product at present mainly contains lamp test and enzyme digestion, and the former loss is higher, and can't be accurately qualitative, and it is long that the latter then exists detection time, the complex operation problem.Therefore study a kind of accurate sensitivity and more simple and rapid detection method has high academic value and application prospect.
Summary of the invention
At the problem that traditional detection method exists, the object of the present invention is to provide provides a kind of sample pre-treatments simple, and Anisakid nematode detection method in easy, quick, sensitive, accurate, the economic marine product.
Purpose of the present invention realizes by following steps:
(1) in the seafood fish body, collects the anisakis stage, after separation and purification, be prepared into somatic antigen;
(2), utilize biological immune technology immune mouse to prepare Anisakid nematode antibody with prepared somatic antigen;
(3) 96 hole polystyrene ELISA Plate are wrapped by comlete antigen in advance and are sealed processing;
(4) sample is extracted separation after, get 50 μ L sample liquid and add in 96 orifice plates, add the antibody diluent of 50 μ L working concentrations again, 37 ℃ of reaction 90min take out back phosphate buffer (0.01mol/L, the pH7.4 that contains 0.1% Tween-20, PBS) the vibration washing is 3 times, each 5min;
(5) get the ELIAS secondary antibody solution of working concentration, get 100 μ L and join in 96 orifice plates, hatch 90min for 37 ℃, take out the back with the PBS that contains 0.1% Tween-20 (0.01mol/L, pH7.4) the vibration washing is 3 times, each 5min;
(6) add 100 μ L substrate developers (3,3 ', 5,5 '-tetramethyl benzidine, 0.1mg/mL) in 96 orifice plates, hatch 20min in the room temperature dark place, add 50 μ L reaction stop solution (H again
2SO
4, 2mol/L).
(7) replace sample liquid with the extract that does not contain determinand simultaneously, similarity condition reacts the back down as negative control; Somatic antigen titer with series concentration replaces sample liquid, and similarity condition is set up typical curve after the reaction down; Carry out the concentration that Anisakid nematode (in somatic antigen) in the sample to be tested is measured in regretional analysis according to typical curve.
Compare with parasite detection techniques such as existing lamp inspection, enzymic digestions, major advantage of the present invention is:
(1) effectively shortened analysis time, operate more easy fast, ELISA Plate generally can be finished detection after wrapping in advance and being closed processing in 4 hours, obviously be better than the enzymic digestion detection method;
(2) sensitivity and accuracy are higher, can detect the Anisakid nematode of trace from the marine product sample;
(3) assemble the detection kit that formation is easy to carry and operates from now on easily, be used for being fit to the rapid screening processing of a large amount of samples.
Embodiment
Describe the present invention in detail below by specific embodiment.
Embodiment 1: the fast detecting of Anisakid nematode in the marine products fillet
1, the preparation of Anisakid nematode somatic antigen
Utilize enzyme digestion separated and collected Anisakid nematode and utilize morphological observation to carry out kind and identify from fillet, the polypide of isolated Anisakis nematode is cleaned with physiological saline, placed glass homogenizer, 4 ℃ of following homogenate behind the PBS of adding 0.01mol/L, the centrifugal 15min of 9700g gets supernatant.The normal hexane degreasing that adds supernatant 1/2 volume then.Extract under 4 ℃ with the redistilled water dialysed overnight, freeze-drying ,-20 ℃ of preservations.
2, the preparation of specific antibody and purifying
Choose 20 of female BALB/C mice in healthy 6-8 age in week, adopt the method for lumbar injection to carry out immunity test, only 0.3mg/ (with protein refractometer), every the 10d immunity once, continuous immunity 5 times is plucked eyeball behind the last immune 7d and is got blood, the centrifugal 30min of 12000g, get supernatant ,-80 ℃ of preservations are standby.
Getting column volume is the HiTrap Protein A HP affinity column of 1mL, and (tertiary sodium phosphate of 20mM pH7.0) is crossed the post flushing, and speed is 1mL/min with the binding buffer liquid of 10 times of column volumes.Get Anisakid nematode antiserum 1mL then,, cross post, 1mL/min with the dilution of 15mL binding buffer liquid; Cross the post flushing with the binding buffer liquid of 10 times of column volumes once more afterwards, to remove unconjugated albumen; (citric acid of 0.1mol/L pH3.6) is crossed the albumen of post elution of bound to the post, and (1mol/L, test tube pH9.0) is collected eluent to fill 300 μ L Tris-HCl with the eluent of three times of column volumes.Eluent under 4 ℃ with the redistilled water dialysed overnight.And being concentrated into 1mL with Macrogol 2000 0, the Bradford method is measured protein content.-80 ℃ of preservations are standby.
3, the bag of ELISA Plate quilt and sealing
With carbonate buffer solution (0.05mol/L, pH 9.6, CBS) dissolving Anisakid nematode somatic antigen (2 μ g/mL) is added to (every hole 100 μ L) in the 96 hole ELISA Plate, 4 ℃ are spent the night.Then with contain 0.1%Tween-20 PBS (PBST) fully washing will be filled with 1% bovine serum albumin(BSA) (BSA is dissolved in PBST) in each hole of ELISA Plate for pH=7.4,0.01mol/L, and 37 ℃ of sealing 1.5h down pat dry after washing three times.
4, mark-on sample pre-treatments
Get wall pollack and sole as the mark-on object, respectively with 0.5,1, article 2, Anisakis nematode and puppet newly the ascarid line belong to larva and join in the 100g flesh of fish, according to the abundant homogenate of ratio of the flesh of fish: PBS (g/mL)=1: 7, after continuing to stir 10 minutes with magnetic stirring apparatus, homogenate is with the centrifugal 15min of 9000r/min, supernatant is diluted to 700mL through the degreasing of n-normal hexane, and 4 ℃ of preservations are standby.
5, mark-on sample detection
At first carry out the chessboard titration experiments, antagonist solution and commercialization ELIAS secondary antibody (sheep anti-mouse antibody) solution suitably dilute to reach the requirement of working concentration as a result according to it; Get the antigen titer (0,100,200,400 of 50 μ L sample liquid and series concentration respectively, 600,800,1000,2000, unit is ng/mL, is dissolved in corresponding blank flesh of fish extract respectively), join in the micropore separately, titer and sample are all done 3 parallel laboratory tests; Add 50 μ L antibody-solutions in each micropore, hatched 90 minutes for 37 ℃; Throw away the liquid in the hole, fully wash 3 times with PBST; Add 100 μ L ELIAS secondary antibody solution, hatched 90 minutes for 37 ℃, throw away the liquid in the hole, with PBST washing 3 times; Add 3,3 of the new preparation of 100 μ L ', 5,5 '-tetramethyl benzidine (TMB) substrate (0.1mg/mL) in micropore, hatched 20 minutes in the room temperature dark place; Add 50 μ L H
2SO
4(2mol/L) stopped reaction in each micropore, with add first of antibody do not classify as blank with microplate reader at 450nm place measurement light absorption value.
Experiment finishes the testing result of back according to the antigen titer, with the concentration of Anisakid nematode somatic antigen (with the soluble protein densimeter, ng/mL) and light absorption value drawing standard curve, according to the concentration (in somatic antigen) of Anisakid nematode in the standard curve determination sample, contain soluble protein 300 μ g calculate recovery rates and carry out the calculating of precision according to every nematode (7mg).
Testing result is as shown in table 1.Newly the ascarid line is when to belong to nematode be 0.5-2 bar/100g flesh of fish when adding Anisakid nematode and puppet, and two recovery that belong to nematodes reach 65.2%-99% and 71.9%-88.1% respectively, and the coefficient of variation is less than 15%, and along with the increase of addition, the recovery has trend of rising.This shows the main performance of this method such as the requirement that accuracy, sensitivity and precision can satisfy actual detected fully; Obviously be less than the enzymic digestion detection technique detection time, operate comparatively easyly, can carry out the detection of a plurality of samples simultaneously on 1 ELISA Plate, the qualitative rapid screening that is very suitable for a large amount of samples detects.
Table 1 mark-on test findings (n=3)
Claims (5)
1. method of utilizing immune reagent kit to detect Anisakid nematode in the marine product is characterized in that may further comprise the steps:
(1) collection separates Anisakid nematode and prepares somatic antigen from seafood fish;
(2) utilize the also specific antibody of purifying Anisakid nematode of biological immune technology preparation;
(3) with the somatic antigen bag by 96 hole ELISA Plate and seal processing;
(4) Anisakid nematode in the sample to be tested is carried out separation and Extraction;
(5) antibody-solutions and the sample extracting solution with working concentration adds in 96 orifice plates simultaneously, reacts according to the step and the method for conventional indirect competitive enzyme-linked immunosorbent (ELISA), finally utilizes microplate reader to measure the OD450 value;
(6) replace sample liquid with the extract that does not contain determinand simultaneously, similarity condition reacts the back down as negative control; Somatic antigen titer with series concentration replaces sample liquid, and similarity condition is set up typical curve after the reaction down;
(7) sample and negative control compare, and cause that the light absorption value reduction promptly is judged as positive sample more than 10%, promptly contain Anisakid nematode to be measured, otherwise negative sample; And carry out the concentration that Anisakid nematode (in somatic antigen) in the sample to be tested is measured in regretional analysis according to the typical curve of being set up.
2. according to right 1 described detection method, it is characterized in that the titer (3) that at first prepares the somatic antigen of Anisakid nematode and form series concentration.
3. according to right 1 described detection method, it is characterized in that at first utilizing the specific polyclonal antibody (4) of biological immune technology preparation at somatic antigen.
4. detection method according to claim 5 is characterized in that Anisakid nematode needs to extract separation in the unknown sample before mensuration.
5. detection method according to claim 1 is characterized in that testing result passes through the light absorption value interpretation, can combined standard curve quantitative measurement sample in the concentration of determinand.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2009100168209A CN101603964A (en) | 2009-07-10 | 2009-07-10 | The enzyme-linked immune detection method of Anisakid nematode in the marine product |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2009100168209A CN101603964A (en) | 2009-07-10 | 2009-07-10 | The enzyme-linked immune detection method of Anisakid nematode in the marine product |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101603964A true CN101603964A (en) | 2009-12-16 |
Family
ID=41469782
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2009100168209A Pending CN101603964A (en) | 2009-07-10 | 2009-07-10 | The enzyme-linked immune detection method of Anisakid nematode in the marine product |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101603964A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102288752A (en) * | 2010-10-09 | 2011-12-21 | 中国疾病预防控制中心寄生虫病预防控制所 | Kit for testing specific antibody with agkistrodon acutus tongue-shaped worm purified antigen |
| CN102879563A (en) * | 2012-10-19 | 2013-01-16 | 中国疾病预防控制中心寄生虫病预防控制所 | Immunochromatography test strip for detecting anisakis disease and preparation method thereof |
| CN103361401A (en) * | 2012-03-30 | 2013-10-23 | 舟山市疾病预防控制中心 | TagMan PCR detection method for anisakis simplexes in marine products |
| CN104561265A (en) * | 2014-11-21 | 2015-04-29 | 深圳市疾病预防控制中心 | Real-time fluorescence PCR detection kit and detection method of anisakis |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1384360A (en) * | 2001-05-02 | 2002-12-11 | 新丰制药株式会社 | Enzyme-linked immunosorbent assay kit for diagnosis of testicular trematodiasis, paragonimiasis, pork-measles disease and sparganosis |
| EP1287025A1 (en) * | 2000-04-19 | 2003-03-05 | Ovita Limited | Sheep lice, bovicola ovis, allergen treatment |
| WO2005058948A1 (en) * | 2003-12-15 | 2005-06-30 | Institut Pasteur | Lsa-5 liver stage and blood stage antigen of plasmodium falciparum, immunogenic composition comprising said antigen, and vaccines against malaria |
| CN101387641A (en) * | 2007-09-11 | 2009-03-18 | 深圳市康百得生物科技有限公司 | Liver fluke rapid immune detection kit for fresh water fishes and detection method |
-
2009
- 2009-07-10 CN CNA2009100168209A patent/CN101603964A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1287025A1 (en) * | 2000-04-19 | 2003-03-05 | Ovita Limited | Sheep lice, bovicola ovis, allergen treatment |
| CN1384360A (en) * | 2001-05-02 | 2002-12-11 | 新丰制药株式会社 | Enzyme-linked immunosorbent assay kit for diagnosis of testicular trematodiasis, paragonimiasis, pork-measles disease and sparganosis |
| WO2005058948A1 (en) * | 2003-12-15 | 2005-06-30 | Institut Pasteur | Lsa-5 liver stage and blood stage antigen of plasmodium falciparum, immunogenic composition comprising said antigen, and vaccines against malaria |
| CN101387641A (en) * | 2007-09-11 | 2009-03-18 | 深圳市康百得生物科技有限公司 | Liver fluke rapid immune detection kit for fresh water fishes and detection method |
Non-Patent Citations (1)
| Title |
|---|
| 全福实等: "异尖线虫四种抗原的酶免测定法检测抗体的比较", 《延边大学医学学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102288752A (en) * | 2010-10-09 | 2011-12-21 | 中国疾病预防控制中心寄生虫病预防控制所 | Kit for testing specific antibody with agkistrodon acutus tongue-shaped worm purified antigen |
| CN103361401A (en) * | 2012-03-30 | 2013-10-23 | 舟山市疾病预防控制中心 | TagMan PCR detection method for anisakis simplexes in marine products |
| CN102879563A (en) * | 2012-10-19 | 2013-01-16 | 中国疾病预防控制中心寄生虫病预防控制所 | Immunochromatography test strip for detecting anisakis disease and preparation method thereof |
| CN104561265A (en) * | 2014-11-21 | 2015-04-29 | 深圳市疾病预防控制中心 | Real-time fluorescence PCR detection kit and detection method of anisakis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102955031B (en) | Enzyme-linked immunosorbent assay kit for detecting aflatoxin B1-containing medicine and application for same | |
| CN104101702B (en) | A kind of indirect competitive enzyme-linked immunosorbent detection method of tetrabromobisphenol A | |
| WO2011057025A3 (en) | Methods and devices to enhance sensitivity and evaluate sample adequacy and reagent reactivity in rapid lateral flow immunoassays | |
| CN103059135A (en) | A specific antibody of major royal jelly protein MRJP1 and a preparation method thereof and Elisa quantitative detection thereof | |
| CN104076154A (en) | Enzyme linked immunosorbent assay kit detecting folic acid and application thereof | |
| CN101196521A (en) | Indirectly racing ELISA detecting method for cadmium ion | |
| CN101603964A (en) | The enzyme-linked immune detection method of Anisakid nematode in the marine product | |
| CN105388296A (en) | Method for detecting tropomyosin by means of homologous epitope peptide antibody | |
| CN101413955A (en) | ELISA test box for detecting zearalenone and preparing and detecting method thereof | |
| RU2013138482A (en) | METHODS AND DEVICE FOR DETECTING GLUTEN SENSITIVITY AND ITS DIFFERENTIATION WITH CELIACIA | |
| Anfossi et al. | Mycotoxins in food and feed: extraction, analysis and emerging technologies for rapid and on-field detection | |
| CN101571540B (en) | Enzyme-linked immunosorbent kit for inspecting porcine immunoglobulin G and application thereof | |
| CN101871936A (en) | Rhodamine B ELISA detection method | |
| CN101893636B (en) | Enzyme-linked immunosorbent assay method for egg allergen ovalbumin in foods | |
| CN104374907B (en) | A kind of indirect competitive enzyme-linked aptamers detects the method for Determination of oxytetracycline residues | |
| CN101196520A (en) | Indirectly racing ELISA detecting method for gonyatoxine GTX2,3 | |
| CN102081094B (en) | Method for detecting patulin and special enzyme linked immunosorbent assay kit thereof | |
| CN105693858A (en) | Preparing method and detecting method for MRJR1 antibody pairs in honey and kit | |
| CN104387467A (en) | Detection kit and detection paper for Beta-adrenergic receptor stimulant multiresidue detection | |
| CN104311659A (en) | Multi-cluster antigen and wide-spectrum specific antibody of beta-adrenoceptor agonists and application of multi-cluster antigen and wide-spectrum specific antibody | |
| CN101692086A (en) | Enzyme-linked immunoassay kit for detecting dienestrol by adopting indirect competition method | |
| CN105301244B (en) | Detect enzyme linked immunological kit and its application of acid orange | |
| CN106483300A (en) | A kind of colloidal gold immuno-chromatography test paper strip of detection Furaxone metabolite and preparation method and application | |
| Torsson et al. | Filter paper is a simple and cost-effective transport medium for serological diagnosis of Peste des petits ruminants | |
| CN102768282B (en) | Preparation and detection methods of kit for detecting chub vitellogenin (VTG) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20091216 |